title: Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.
abstract: Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.
authors.affiliation: Aix Marseille Université, CNRS, Centrale Marseille, Institut Fresnel, UMR 7249, F-13013 Marseille, France.
authors.forename: Alla
authors.initials: A
authors.lastname: Kress
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.forename: Hubert
authors.initials: H
authors.lastname: Ranchon
authors.forename: Julien
authors.initials: J
authors.lastname: Savatier
authors.forename: Hervé
authors.initials: H
authors.lastname: Rigneault
authors.forename: Patrick
authors.initials: P
authors.lastname: Ferrand
authors.forename: Sophie
authors.initials: S
authors.lastname: Brasselet
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D002784:Cholesterol